Laboratory of Protein structure, stability and folding of proteins, Institute of Cytology RAS, 194064 St. Petersburg, Russia.
J Phys Chem B. 2011 Jul 28;115(29):9022-32. doi: 10.1021/jp204555h. Epub 2011 Jun 29.
The galactose/glucose-binding protein from E. coli (GGBP) is a 32 kDa protein possessing the typical two-domains structure of the ligand-binding proteins family. GGBP is characterized by low dissociation constant values with respect to glucose binding, displaying an affinity constant for glucose in micromolar range. This feature makes GGBP unsuitable as a sensitive probe for continuous glucose monitoring in blood of diabetic patients. In this work we designed, produced, and characterized two mutant forms of GGBP carrying the following amino acid substitutions in the active center of the protein: W183A or F16A. The two mutant GGBP forms retained a globular structure similar to that of the wild-type GGBP and displayed an affinity for glucose lower than the wild-type GGBP. A deep inspection of the entire set of the obtained results pointed out that the N- and C-terminal domains of GGBP-W183A in the absence of glucose have a stability lower than that of the wild-type protein. In the presence of glucose, the two domains of GGBP-W183A were tightly bound, making the protein structure more stable to the action of denaturing agents. On the contrary, the mutant form GGBP-F16A possesses a very restricted structural stability both in the absence and in the presence of glucose. In this work the role of Phe 16 and W 183 are discussed with regard to the structural and functional features of GGBP. In addition, some general guidelines are reported for the design of a novel glucose biosensor based on the use of GGBP.
大肠杆菌的半乳糖/葡萄糖结合蛋白(GGBP)是一种 32kDa 的蛋白质,具有配体结合蛋白家族的典型双结构域结构。GGBP 与葡萄糖结合的解离常数值较低,对葡萄糖的亲和力常数处于微摩尔范围内。这一特性使得 GGBP 不适合作为糖尿病患者血液中连续血糖监测的敏感探针。在这项工作中,我们设计、生产并表征了两种突变形式的 GGBP,它们在蛋白质的活性中心携带以下氨基酸取代:W183A 或 F16A。两种突变 GGBP 形式保留了与野生型 GGBP 相似的球状结构,并表现出低于野生型 GGBP 的葡萄糖亲和力。对整个获得结果的深入检查表明,在没有葡萄糖的情况下,GGBP-W183A 的 N 和 C 末端结构域的稳定性低于野生型蛋白。在葡萄糖存在的情况下,GGBP-W183A 的两个结构域紧密结合,使蛋白质结构对变性剂的作用更稳定。相反,突变形式的 GGBP-F16A 无论在有无葡萄糖的情况下,其结构稳定性都非常有限。在这项工作中,讨论了 Phe16 和 W183 在 GGBP 的结构和功能特征方面的作用。此外,还报告了一些基于 GGBP 使用的新型葡萄糖生物传感器设计的一般准则。